Electron spin echo envelope modulation (ESEEM) experiments performed on the Rieske Fe-S clusters of the cytochrome b6f complex of spinach chloroplasts and of the cytochrome bc1 complexes of Rhodospirillum rubrum, Rhodobacter sphaeroides R-26, and bovine heart mitochondria show modulation components resulting from two distinct classes of 14N ligands. At the g = 1.92 region of the Rieske EPR spectrum of the cytochrome b6f complex, the measured hyperfine couplings for the two classes of coupled nitrogens are A1 = 4.6 MHz and A2 = 3.8 MHz. Similar couplings are observed for the Rieske centers in the three cytochrome bc1 complexes. These ESEEM results indicate a nitrogen coordination environment for these Rieske Fe-S centers that is similar to that of the Fe-S cluster of a bacterial dioxygenase enzyme with two coordinated histidine ligands [Gurbiel, R. J., Batie, C. J., Sivaraja, M., True, A. E., Fee, J. A., Hoffman, B. M., & Ballou, D. P. (1989) Biochemistry 28, 4861-4871]. The Rieske Fe-S cluster lacks modulation components from a weakly coupled peptide nitrogen observed in water-soluble spinach ferredoxin. Treatment with the quinone analogue inhibitor DBMIB causes a shift in the Rieske EPR spectrum to g = 1.95 with no alteration in the magnetic coupling to the two nitrogen atoms. However, the ESEEM pattern of the DBMIB-altered Rieske EPR signal shows evidence of an additional weakly coupled nitrogen similar to that observed in the spinach ferredoxin ESEEM patterns.

Plastocyanin has been covalently cross-linked to photosystem I (PSI) by using a water-soluble cross-linker, N-ethyl-3-[3-(dimethylamino)propyl]carbodiimide. The cross-linking reaction is light stimulated and results in the disappearance of a single 19-kDa subunit of PSI with the formation of a new protein-staining component of 31 kDa. The new product at 31 kDa reacts with both plastocyanin and 19-kDa subunit antibodies. Carboxyl group modified plastocyanin does not form a cross-linked product with PSI, implying that the negatively charged surface-exposed groups on plastocyanin are necessary to stabilize binding. These results demonstrate a specific interaction of plastocyanin with PSI and further implicate a specific protein to which plastocyanin binds to facilitate electron transfer to the P700 reaction center.

The chlorophyll a/b‐protein complex (LHCPI) associated with Photosystem I (PS I) has been isolated from spinach thylakoids and further fractionated into two chlorophyll‐containing complexes by sucrose gradient centrifugation. The lighter fraction contains two polypeptides with relative molecular masses of 23 and 22 kDa and has been designated as LHCPIa. The denser fraction is enriched in a 20 kDa polypeptide and has been named LHCPIb. Both fractions have a chlorophyll a/b ratio of 3.5 ± 0.5. The absorption spectra and 77 K fluorescence emission spectra of the fractions show distinct characteristics with LHCPIb having a fluorescence maximum at 730 nm at 77 K while LHCPIa shows a maximum at 680 nm. The optical activities of the chlorophyll a/b complexes and the antenna‐depleted PS I have been examined by circular dichroism (CD) in the near‐UV and visible regions of the spectrum. All the LHCPI complexes show strong CD signals at 648, 485 and 340 nm which are absent in the antenna‐depleted PS I complex.

We have used a cross-linking approach to study the interaction of ferredoxin (Fd) with photosystem I (PSI). The cross-linking reagent carbodiimide was found to cross-link spinach Fd to a 22 kilodalton subunit of PSI in both isolated spinach (Spinacia oleracea) PSI complexes and spinach thylakoid membranes.The product had an apparent molecular weight of 38 kilodaltons on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and was identified as a cross-linked product using specific antibodies to Fd and the 22 kilodalton subunit. In both a native PSI complex (200 Chl/P700) and a PSI core complex (100 Chl/P700), a second cross-linked product at 36 kilodaltons was seen. The latter cross-reacted with an antibody to Fd but did not cross-react with antibodies directed against the 24.3, 22, 19, 17.3 or 8.5 kilodalton, or psaC subunits of PSI. Its composition remains to be determined. In thylakoids only the 38 kilodalton product was observed along with a cross-linked complex of Fd and Fd:NADP' reductase.Chloroplast thylakoid membranes contain three integral membrane protein complexes which cooperate in the transfer of electrons from water to NADP. These are PSI, PSII, and the Cyt b6-f complex. In this noncyclic electron transport chain, PSI catalyzes light-induced electron transport from reduced plastocyanin to Fd (10).The PSI complex contains P700 as its reaction center Chl and a series of electron acceptors, Ao, A,, Fe-Sx, Fe-SA, and Fe-SB, which accept the electron lost from the reaction center Chl upon photooxidation (10,16 plexes. This cross-linker has previously been used to cross-link Fd to Fd:NADP+ reductase (20) and to cross-link plastocyanin to Cyt f (3). In this study, cross-linked products have been identified by immunoblotting with specific antibodies. Using this approach, a 22 kD subunit ofPSI has been identified as a possible Fd-binding protein in the PSI complex. MATERIALS AND METHODSThylakoid and PSI Preparation. Spinach (Spinacia oleracea) was grown hydroponically in a greenhouse. Washed, deribbed leaves were homogenized in blending buffer (50 mM Tris-HCl [pH 7.8], 0.3 M sucrose, 10 mM NaCl, 5 mM MgCl2). The homogenate was passed through filtering silk and centrifuged for 3 min at 3000 g. The pellets were resuspended in 10 mM MOPS buffer (pH 6.5) for use in cross-linking, or in blending buffer for activity assays. PSI-200 was prepared by the method of Mullet et aL (12). PSI-100 was prepared as described by Ortiz et al. (14). The preparations used in this work contained 185 Chl/P700 and 120 Chl/P700, respectively.Cross-Linking. Ferredoxin was cross-linked to thylakoids as described by Merati and Zanetti (I 1) with the following changes. The cross-linking reaction was carried out in 10 mM MOPS buffer (pH 6.5), 2 mM MgCl2 with 2.5 mm EDC. After quenching the reaction, blending buffer was used to dilute the membranes, which were then pelleted and resuspended in blending buffer.PSI-200 (0.5 mg Chl/mL) was incubated in the presence or absence of 30 jM Fd and 1 mm EDC in the MOPS-MgCl2 buffer f...

ABSTRACICultures of the blue-reen alga, Aphanocapsa, were grown under irolimiting conditions and changes in concentration of redox components of the photosynthetic electon transport chain, patclarly iron-sulfur centers, were monitored by spectoscopic methods. A moderate iron depletion (1/10 of the normal concentration) had little effect on photosynthetic electron transport reactions and growth. Nevertheless, the amount of membrane-bound non-heme iron decreased sharply, and ferredoxin was nearly totally replaced by a flavin-containing protein, flavodoxin. Severe iron-deficiency (1/100 of the normal concentration) was accompanied by growth inhibition and decreased rates of photosynthetic eletr flow. The Photosystem I reaction center was most affected by iron depletion as evidenced by a decrease in the amounts of iron-sulfur centers A, B, and X. However, formation of other redox proteins, even those that do not contain iron, was also inhibited by severe iron deficiency.Various Fe-containing proteins are involved in electron transfer processes ( 17) in energy-transducing membranes. In the photosynthetic electron transport chain, heme-containing proteins (cytochromes) and non-heme Fe-containing proteins (Fd-like proteins) can be found. Except for the plastidal 'Rieske' Fe-S center, which is a component of the Cyt b6-fcomplex (5), other Fe-S centers are located in the electron acceptor complex of PSI (9).In contrast to mitochondria, where Fe deficiency has been reported to result in a decrease of respiratory Fe-S centers and respiratory function (8, 13), a detailed study of the effect of limited Fe supply on plastidal Fe-S centers as well as on photosynthetic electron transport has not been made. There are reports of decrease in P700, the reaction center Chl of PSI and c-type Cyt in algae grown at low Fe concentrations (2, 12) but there are no reports of the effect of such growth conditions on membranebound Fe-S centers. In the present study, we have investigated the effect of Fe deficiency on the formation of Fe-S centers and other redox components of PSI and on the plastidal Cyt b6-f complex in the blue-green alga Aphanocapsa. The effect of Fe deficiency on partial reactions of the photosynthetic electron transport chain was also considered.